The invention generally relates to human biology discoveries and therapeutic and diagnostic compositions and methods based thereon. More particularly, the invention relates to human homeobox gene ventx and its control of macrophage terminal differentiation and activation, and related therapeutic and diagnostic compositions and methods of use, in particular in connection with inflammatory diseases.
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1. A method for identifying a compound which regulates ventx expression, the method comprising:
(a) providing a cell that contains an exogenous reporter gene operably linked to a ventx promoter, the ventx promoter being 2.8 KB in length cloned with the primers: 5′-CAGCCGAGTCTCACTCTGTC-3′ (SEQ ID NO: 1) and 5′-CAAAGCTGGAGAGCTGCTGC-3′ (SEQ ID NO: 2);
(b) contacting the cell with a candidate compound; and
(c) measuring the reporter gene activity,
wherein the ventx promoter is active in the cell and the candidate compound is a regulator of ventx expression if the measured reporter gene activity is modulated as compared to a baseline activity of the reporter gene measured in the absence of the candidate compound.
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This application is the national stage of International Application No. PCT/US2012/041806, filed Jun. 10, 2012, which claims the benefit of U.S. Provisional Application No. 61/495,849, filed Jun. 10, 2011, the entire content of which is expressly incorporated herein by reference.
The invention generally relates to human biology discoveries and therapeutic and diagnostic compositions and methods based thereon. More particularly, the invention relates to human homeobox gene VentX and its control of macrophage terminal differentiation and activation, and related therapeutic and diagnostic compositions and methods of use.
Tissue macrophages are cells produced by the differentiation of monocytes in tissues in response to microenvironmental factors such as M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte macrophage colony-stimulating factor), or IL3 during extravascularization. (Serbina, et al. 2008 Annu Rev Immunol 26:421-452.) Macrophages play critical roles in both innate and adaptive immunity in virtually all tissues. (Auffray, et al. 2009 Annu Rev Immunol 27:669-692; Martinez, et al. 2008 Front Biosci 13:453-461; Mosser, et al. 2008 Nat Rev Immunol 8:958-969.)
The process of monocyte to macrophage terminal differentiation remains a subject of extensive investigation in the contexts of immune defense against pathogen invasion, pathogenesis of autoimmune and inflammatory diseases, and carcinogenesis of hematopoietic and other malignancies. (Tenen 2003 Nat Rev Cancer 3:89-101.) Upon differentiation, the function of macrophages can be further activated by extracellular signals and displays diverse patterns depending upon the cytokines and microbial products present in the microenvironment.
Macrophage activation has been classified into a classical pathway and an alternative pathway. In response to Th1 cytokines, such as interferon γ (IFN-γ) and lipopolysaccharide (LPS), macrophages display a classical activation phenotype and produce mainly pro-inflammatory cytokines. The Jak/Stat and AP-1/NFκB signaling pathways have been shown to play critical roles in classical activation of macrophages. Alternatively, macrophages can be activated by Th2 cytokines, such as IL4 or IL13, and exhibit distinct functions with anti-inflammatory and tissue repair properties. (Martinez, et al. 2008 Front Biosci 13:453-461; Mosser, et al. 2008 Nat Rev Immunol 8:958-969; Gordon 2003 Nat Rev Immunol 3:23-35; Schroder, et al. 2006 Immunobiology 211:511-524.)
The common myeloid progenitor cells are the bone marrow precursors of monocytes and macrophages. It is generally accepted that monocyte and macrophage development occurs by changes of transcriptional programs in a stepwise manner. (Friedman 2007 Oncogene 26:6816-6828; Friedman 2002 Oncogene 21:3377-3390; Valledor, et al. 1998 J Leukoc Biol 63:405-417; Tenen, et al. 1997 Blood 90:489-519.) Genetic studies with knockout mice have revealed the important roles of transcription factors such as PU.1 and C/EBPα in monocyte/macrophage lineage commitment. (Yeamans, et al. 2007 Blood 110:3136-3142; Scott, et al. 1994 Science 265:1573-1577; McKercher, et al. 1996 Embo J 15:5647-5658.)
Recently, global transcriptome analysis revealed profound changes in gene expression during monocyte to macrophage terminal differentiation. (Liu, et al. 2008 Immunol Lett 117:70-80; Martinez, et al. 2006 J Immunol 177:7303-7311.) Previous studies on human monocyte to macrophage differentiation have mainly relied on myeloid progenitor cell lines like U937 and THP-1. (Lu, et al. 2001 J Biol Chem 276:45491-45496; Chang, et al. 2000 Nat Immunol 1:169-176; Liu, et al. 1996 Genes Dev 10:142-153.) The key transcriptional mechanism controlling primary human monocyte to macrophage differentiation remains poorly defined.
Developmental modeling is informative in defining genes and pathways involved in host defense and immune regulation. Using methods of reverse genetics, it was recently demonstrated that VentX, a human homologue of the Xenopus homeobox transcriptional factor Xom, is a LEF/TCF-associated Wnt repressor and a putative tumor suppressor. (Gao, et al. 2010 Cancer Res 70:202-211; Gao, et al. 2007 Cell Res 17:345-356; U.S. Pat. No. 7,994,126, expressly incorporated herein by reference for all purposes; WO/2011/00894, PCT/US2010/042126, expressly incorporated herein by reference for all purposes.) Also shown was that VentX trans-activates p53/p21 and p16ink4a/Rb pathways to regulate senescence in tumor cells. (Wu, et al. 2011 J Biol Chem 286:12693-12701) VentX is predominantly expressed in hematopoietic cells and highly conserved in primates. However, researches have failed to identify the murine homologue of VentX in the current mouse genome database (20, 23, 24). (Gao, et al. 2010 Cancer Res 70:202-211; Ku, et al. 2006 J Biol Chem 281:5277-5287; Rawat, et al. 2010 Proc Natl Acad Sci USA 107:16946-16951.)
Therefore, a continued need exists for better understanding of the role of VentX, in particular in relation to microphage differentiation and activation, and therapeutic and/or diagnostic applications based therefrom.
The invention is based, in part, on the discovery that VentX plays an essential role in human primary monocyte to macrophage terminal differentiation and is required for optimal pro-inflammatory response during macrophage classical activation. For instance, the expression level of VentX correlates positively with the expression levels of several pro-inflammatory cytokines, indicating a role for VentX in the pathogenesis of inflammatory diseases.
In one aspect, the invention generally relates to a method for treating an inflammatory disease, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an biological or chemical agent that exerts a modulating effect of human homeobox gene VentX.
In another aspect, the invention generally relates to a pharmaceutical composition comprising an biological or chemical agent that exerts a modulating effect of human homeobox gene VentX.
In yet another aspect, the invention generally relates to a method for identifying a compound which regulates the VentX expression. The method includes: (a) providing a cell comprising a polynuceotide comprising VentX promoter; (b) contacting the cell with a candidate compound; and (c) measuring the activity of VentX expression. In certain preferred embodiments, the VentX promoter sequence is of 2.8 KB, cloned with the primers: 5′-CAGCCGAGTCTCACTCTGTC-3′ (SEQ ID NO:1) and 5′-CAAAGCTGGAGAGCTGCTGC-3′ (SEQ ID NO:2), wherein the promoter sequence is placed in front of a luciferase gene to create a construct for a promoter-luciferase assay.
The definitions below are provided as summaries of concepts that are commonly understood by one of ordinary skill in the relevant art and are provided for the purposes of understanding of the subject matter disclosed herein. The definitions are not meant to be limitations of the invention or claims herein.
As used herein, the term “antibody” refers to molecules which are capable of binding an epitope or antigenic determinant. The term is meant to include whole antibodies and antigen-binding fragments thereof, including single-chain antibodies. The antibodies can be from any animal origin. Preferably, the antibodies are mammalian, e.g., human, murine, rabbit, goat, guinea pig, camel, horse and the like, or other suitable animals. Antibodies may recognize polypeptide or polynucleotide antigens. The term includes active fragments, including for example, an antigen binding fragment of an immunoglobulin, a variable and/or constant region of a heavy chain, a variable and/or constant region of a light chain, a complementarity determining region (cdr), and a framework region. The terms include polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies, hybrid antibody molecules, F(ab)2 and F(ab) fragments; Fv molecules (for example, noncovalent heterodimers), dimeric and trimeric antibody fragment constructs; minibodies, humanized antibody molecules, and any functional fragments obtained from such molecules, wherein such fragments retain specific binding. The use of the singular terms “a” or “an” or “the” antibody are not meant to be limited to a single antibody when it is clear that more than one antibody is present in the composition or preparation. In addition, unless indicated otherwise, the singular term for “antibody” may include a collection of antibodies that are not necessarily heterogenous in their structures or specificities.
As used herein, the term “humanized” antibodies refer to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise either complete variable domains fused onto constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domains. Antigen binding sites may be wild type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulin more closely. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody which contains all six CDRs from the mouse antibodies). Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered with respect to the original antibody.
The term “binds specifically,” in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific epitope. Hence, an antibody that binds specifically to one epitope (a “first epitope”) and not to another (a “second epitope”) is a “specific antibody.” An antibody specific to a first epitope may cross react with and bind to a second epitope if the two epitopes share homology or other similarity. The term “binds specifically,” in the context of a polynucleotide, refers to hybridization under stringent conditions. Conditions that increase stringency of both DNA/DNA and DNA/RNA hybridization reactions are widely known and published in the art (Curr. Prot. Molec. Biol., John Wiley & Sons (2001)).
As used herein, the term “antigen” refers to a molecule capable of being bound by an antibody. An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a Th cell epitope and is given in adjuvant. An antigen can have one or more epitopes (B- and/or T-cell epitopes). The specific reaction referred to above is meant to indicate that the antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens. Antigens as used herein may also be mixtures of several individual antigens.
As used herein, the term “epitope” refers to basic element or smallest unit of recognition by an individual antibody or T-cell receptor, and thus the particular domain, region or molecular structure to which said antibody or T-cell receptor binds. An antigen may consist of numerous epitopes while a hapten, typically, may possess few epitopes.
As used herein, the term “nucleic acid molecule,” “nucleotide,” “oligonucleotide,” “polynucleotide,” and “nucleic acid” are used interchangeably herein to refer to polymeric forms of nucleotides of any length. They can include both double- and single-stranded sequences and include, but are not limited to, cDNA from viral, prokaryotic, and eukaryotic sources; mRNA; genomic DNA sequences from viral (e.g., DNA viruses and retroviruses) or prokaryotic sources; RNAi; cRNA; antisense molecules; ribozymes; and synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.
As used herein, a “complementary” nucleotide sequence acid molecule is a one that is comprised of its base pair complements. Deoxyribonucleotides with the base adenine are complementary to those with the base thymidine, and deoxyribonucleotides with the base thymidine are complementary to those with the base adenine. Deoxyribonucleotides with the base cytosine are complementary to those with the base guanine, and deoxyribonucleotides with the base guanine are complementary to those with the base cytosine. Ribonucleotides with the base adenine are complementary to those with the base uracil, and deoxyribonucleotides with the base uracil are complementary to those with the base adenine. Ribonucleotides with the base cytosine are complementary to those with the base guanine, and deoxyribonucleotides with the base guanine are complementary to those with the base cytosine.
As used herein, the term “promoter” refers to a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of a downstream (3′ direction) coding sequence operably linked thereto. For purposes of the present invention, a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence may be a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Promoters include those that are naturally contiguous to a nucleic acid molecule and those that are not naturally contiguous to a nucleic acid molecule. Additionally, the term “promoter” includes inducible promoters, conditionally active promoters such as a cre-lox promoter, constitutive promoters, and tissue specific promoters.
As used herein, the term “transfected” means possessing introduced DNA or RNA, with or without the use of any accompanying facilitating agents such as lipofectamine. Methods for transfection that are known in the art include calcium phosphate transfection, DEAE dextran transfection, protoplast fusion, electroporation, and lipofection.
As used herein, the term “expression of a nucleic acid molecule” refers to the conversion of the information contained in the nucleic acid molecule into a gene product. The gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a peptide or polypeptide produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing; and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
As used herein, the term “host cell” refers to an individual cell or a cell culture that can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide(s). Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell that comprises a recombinant vector of the invention may be called a “recombinant host cell.”
As used herein, the term an “isolated” or “substantially isolated” molecule (such as a polypeptide or polynucleotide) is one that has been manipulated to exist in a higher concentration than in nature or has been removed from its native environment. For example, a subject antibody is isolated, purified, substantially isolated, or substantially purified when at least 10%, or 20%, or 40%, or 50%, or 70%, or 90% of non-subject-antibody materials with which it is associated in nature have been removed. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated.” Further, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA and RNA molecules. Isolated nucleic acid molecules further include synthetically produced molecules. Additionally, vector molecules contained in recombinant host cells are also isolated. Thus, not all “isolated” molecules need be “purified.”
As used herein, the term “purified” when used in reference to a molecule, it means that the concentration of the molecule being purified has been increased relative to molecules associated with it in its natural environment, or environment in which it was produced, found or synthesized. Naturally associated molecules include proteins, nucleic acids, lipids and sugars but generally do not include water, buffers, and reagents added to maintain the integrity or facilitate the purification of the molecule being purified. According to this definition, a substance may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% pure when considered relative to its contaminants.
As used herein, the term “biologically active” entity, or an entity having “biological activity,” is one having structural, regulatory, or biochemical functions of a naturally occurring molecule or any function related to or associated with a metabolic or physiological process. Biologically active polynucleotide fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a polynucleotide of the present invention. The biological activity can include an improved desired activity, or a decreased undesirable activity. For example, an entity demonstrates biological activity when it participates in a molecular interaction with another molecule, such as hybridization, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, when it has diagnostic and/or prognostic value in determining the presence of a molecule, such as a biologically active fragment of a polynucleotide that can, for example, be detected as unique for the polynucleotide molecule, or that can be used as a primer in a polymerase chain reaction. A biologically active polypeptide or fragment thereof includes one that can participate in a biological reaction.
As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a living animal, including a human and a non-human animal. The subject may, for example, be an organism possessing immune cells capable of responding to antigenic stimulation, and stimulatory and inhibitory signal transduction through cell surface receptor binding. The subject may be a mammal, such as a human or non-human mammal, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. The term “subject” does not preclude individuals that are entirely normal with respect to a disease, or normal in all respects.
As used herein, a “patient sample” is any biological specimen derived from a patient. The term includes, but is not limited to, biological fluids such as blood, serum, plasma, urine, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid, lavage fluid, semen, and other liquid samples, as well as cell and tissues of biological origin. The term also includes cells or cells derived therefrom and the progeny thereof, including cells in culture, cell supernatants, and cell lysates. It further includes organ or tissue culture-derived fluids, tissue biopsy samples, tumor biopsy samples, stool samples, and fluids extracted from physiological tissues, as well as cells dissociated from solid tissues, tissue sections, and cell lysates. This definition encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides or polypeptides. Also included in the term are derivatives and fractions of patient samples. A patient sample may be used in a diagnostic, prognostic, or other monitoring assay.
As used herein, the term “modulate” refers to the production, either directly or indirectly, of an increase or a decrease, a stimulation, inhibition, interference, or blockage in a measured activity when compared to a suitable control. A “modulator” of a polypeptide or polynucleotide or an “agent” are terms used interchangeably herein to refer to a substance that affects, for example, increases, decreases, stimulates, inhibits, interferes with, or blocks a measured activity of the polypeptide or polynucleotide, when compared to a suitable control.
As used herein, the terms “disease” or “disorder” refer to a pathological condition, for example, one that can be identified by symptoms or other identifying factors as diverging from a healthy or a normal state. The term “disease” includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.
As used herein, the term “inflammatory condition(s)” refers to the group of conditions including, rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, allergic airway disease (e.g., asthma, rhinitis), inflammatory bowel diseases (e.g., Crohn's disease, colitis), endotoxin-driven disease states (e.g., complications after bypass surgery or chronic endotoxin states contributing to e.g. chronic cardiac failure), and related diseases involving cartilage, such as that of the joints. Partcicularly the term refers to rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g., asthma) and inflammatory bowel diseases.
As used herein, the term “autoimmune disease(s)” refers to the group of diseases including obstructive airways disease, including conditions such as COPD, asthma (e.g., intrinsic asthma, extrinsic asthma, dust asthma, infantily asthma) particularly chronic or inveterate asthma (for example late asthma and airway hyperreponsiveness), bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), multiple sclerosis, type I diabetes mellitus and complications associated therewith, atopic eczema (atopic dermatitis), contact dermatitis and further eczematous dermatitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. Particularly the term refers to COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease.
As used herein the term “proliferative disease(s)” refers to conditions such as cancer (e.g., uterine leiomyosarcoma or prostate cancer), myeloproliferative disorders (e.g., polycythemia vera, essential thrombocytosis and myelofibrosis), leukemia (e.g., acute myeloid leukaemia and acute lymphoblastic leukemia), multiple myeloma, psoriasis, restenosis, sclerodermitis or fibrosis. In particular the term refers to cancer, leukemia, multiple myeloma and psoriasis.
As used herein, the terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, sarcoma, blastoma and leukemia. More particular examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer.
As used herein, the term “tumor” refers to any malignant or neoplastic cell.
As used herein, the term “treatment” covers either prophylactic and/or therapeutic treatments including any administration or application of remedies for disease in a mammal, including a human, and includes inhibiting the disease. It includes arresting disease development and relieving the disease, such as by causing regression or restoring or repairing a lost, missing, or defective function, or stimulating an inefficient process. As used herein, the term “preventing” includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. Treatment and prophylaxis can be administered to an organism, including a human, or to a cell in vivo, in vitro, or ex vivo, and the cell subsequently administered the subject.
As used herein, the term “effective amount” refers to an amount necessary or sufficient to realize a desired biologic effect. An effective amount of the composition would be the amount that achieves this selected result, and such an amount could be determined as a matter of routine by a person skilled in the art. For example, an effective amount for treating an immune system deficiency could be that amount necessary to cause activation of the immune system, resulting in the development of an antigen specific immune response upon exposure to antigen. The term is also synonymous with “sufficient amount.” The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.
As used herein, the term “carrier” refers to a solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of any conventional type. A “pharmaceutically acceptable carrier” refers to a non-toxic “carrier.” A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. Pharmaceutically acceptable carriers can be, for example, vehicles, adjuvants, or diluents.
As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. Furthermore, a “polypeptide” may refer to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental.
As used herein, the term “receptor” refers to proteins or glycoproteins or fragments thereof capable of interacting with another molecule, called the ligand. The ligand may belong to any class of biochemical or chemical compounds. The ligand is usually an extracellular molecule which, upon binding to the receptor, usually initiates a cellular response, such as initiation of a signal transduction pathway. The receptor need not necessarily be a membrane-bound protein.
As used herein, the term “recombinant,” with respect to a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant”, as used with respect to a protein or polypeptide, means a polypeptide produced by expression of a recombinant polynucleotide. The term “recombinant” as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.
As used herein, the phrase “recombinant virus” refers to a virus that is genetically modified by the hand of man. The phrase covers any virus known in the art.
As used herein, the term “vector” refers to an agent (e.g., a plasmid or virus) used to transmit genetic material to a host cell or organism. A vector may be composed of either DNA or RNA.
As used herein, the term “interfering RNA” or “RNAi” or “interfering RNA sequence” refers to double-stranded RNA (i.e., duplex RNA) that is capable of reducing or inhibiting expression of a target gene (i.e., by mediating the degradation of mRNAs which are complementary to the sequence of the interfering RNA) when the interfering RNA is in the same cell as the target gene. Interfering RNA thus refers to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering RNA may have substantial or complete identity to the target gene or may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the interfering RNA can correspond to the full length target gene, or a subsequence thereof. Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length).
As used herein, the term “sample” refers to a sample from a human, animal, or to a research sample, e.g., a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulated material. The “sample” may be tested in vivo, e.g., without removal from the human or animal, or it may be tested in vitro. The sample may be tested after processing, e.g., by histological methods. “Sample” also refers, e.g., to a cell comprising a fluid or tissue sample or a cell separated from a fluid or tissue sample. “Sample” may also refer to a cell, tissue, organ, or fluid that is freshly taken from a human or animal, or to a cell, tissue, organ, or fluid that is processed or stored.
The invention is based, in part, on the discovery that VentX plays an essential role in human primary monocyte to macrophage terminal differentiation and is required for optimal pro-inflammatory response during macrophage classical activation. For instance, the expression level of VentX correlates positively with the expression levels of several pro-inflammatory cytokines, indicating a role for VentX in the pathogenesis of inflammatory diseases.
VentX Regulates Monocyte to Macrophage Terminal Differentiation
The molecular mechanisms underlying monocyte/macrophage development have been extensively investigated for their broad implications in host defense, autoimmunity, inflammatory control, and tissue repair and regeneration (1, 3, 10, 53). (Auffray, et al. 2009 Annu Rev Immunol 27:669-692; Mosser, et al. 2008 Nat Rev Immunol 8:958-969; Valledor, et al. 1998 J Leukoc Biol 63:405-417; Gordon, et al. 2005 Nat Rev Immunol 5:953-964.) Monocytes/macrophages arise from pluripotent hematopoietic stem cells in the bone marrow through multiple stages of concerted expression of signaling molecules and transcriptional factors. (Friedman 2007 Oncogene 26:6816-6828; Valledor, et al. 1998 J Leukoc Biol 63:405-417; Tenen, et al. 1997 Blood 90:489-519.) Extensive genetic studies in knockout mice have identified several transcriptional factors that are important for specification of myeloid progenitor cells and subsequent monocyte/macrophage lineage commitment. (Feinberg, et al. 2007 Embo J 26:4138-4148.) In particular, the Ets family transcriptional factor PU.1 and C/EBP family member C/EBPα represent master regulators of myeloid lineage development. (Tenen, et al. 1997 Blood 90:489-519; Yeamans, et al. 2007 Blood 110:3136-3142; Scott, et al. 1994 Science 265:1573-1577; McKercher, et al. 1996 Embo J 15:5647-5658.)
Nevertheless, it is clear that monocyte/macrophage lineage commitment is distinct from differentiation of monocytes into mature macrophages; the latter is accompanied by marked changes in cell morphology and immune functions and requires a new set of expressed genes. (Liu, et al. 2008 Immunol Lett 117:70-80; Martinez, et al. 2006 J Immunol 177:7303-7311; Imhof, et al. 2004 Nat Rev Immunol 4:432-444; Gordon, et al. 2005 Nat Rev Immunol 5:953-964.) However, little is known about the transcriptional regulation of terminal monocyte to macrophage differentiation. The data suggest that VentX is a key regulator of this process. In the knockdown experiments, 50% to 70% of VentX suppression could be achieved with the siRNA approach, which resulted in a remarkable 80% reduction of CD71 expression (
Among the questions to be answered are the molecular mechanisms of VentX upregulation during monocyte to macrophage differentiation (
Mechanism of Regulation of Macrophage Differentiation by VentX
M-CSF receptor (M-CSFR, also called csflr) is an integral membrane tyrosine kinase encoded by the c-fms proto-oncogene. M-CSFR is expressed in monocytes/macrophages and their progenitors, and is obligatory for macrophage differentiation. (Bourette, et al. 2000 Growth Factors 17:155-166; Pixley, et al. 2004 Trends Cell Biol 14:628-638.) Several cytokines and transcription factors regulate macrophage differentiation via modulating expression of M-CSFR (37, 58, 59). (Shi, et al. 2004 J Clin Invest 114:408-418.) In
VentX Controls Macrophage Pro-Inflammatory Responses
Knockdown of VentX caused downregulation of multiple membrane receptors critical for innate and adaptive immunity, including Toll-like receptor 4, mannose receptor, Fcγ receptor CD64, co-stimulatory molecules CD40, CD80 and CD86, and adhesion molecules CD11b and CD11c. As a consequence, the profoundly phenotypic and functional changes were observed in VentX silenced monocytes/macrophages. Specifically, downregulation of TLR4, CD14, MR and CD64 may contribute to the impaired phagocytotic ability of monocyte/macrophage (61). Aberrant morphogenesis and adherence may relate to the downregulation of adhesion molecules CD11b and CD11c. (Imhof, et al. 2004 Nat Rev Immunol 4:432-444.) Downregulation of co-stimulatory molecules CD40, CD80 and CD86 on membrane may weaken the capability of macrophage to stimulate T cell proliferation (
Consequently, knockdown of VentX rendered macrophages unable to mount optimal pro-inflammatory response upon classical activation (
Thus, dysregulated expression of VentX in macrophages may play a role in the pathogenesis of autoimmune diseases. Moreover, it was found that expression of VentX could be down-regulated by immunosuppressive therapy in SLE/RA patients (
In one aspect, the invention generally relates to a method for treating an inflammatory disease, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an biological or chemical agent that exerts a modulating effect of human homeobox gene VentX.
In certain preferred embodiments, the modulating effect comprises an inhibiting effect.
In certain preferred embodiments, the biological or chemical agent is a polypeptide. For example, the biological or chemical agent may comprise a VentX mutant lacking the homeodomain as a blocking polypeptide. The biological or chemical agent comprises the VentX homeodomain without other activating domain as a blocking polypeptide.
In certain preferred embodiments, the polypeptide may be delivered into cells via a short delivery peptide.
In certain preferred embodiments, the biological or chemical agent is an antibody fragment.
In certain preferred embodiments, the biological or chemical agent is an oligonucleotide, for example, an RNAi.
In another aspect, the invention generally relates to a pharmaceutical composition comprising an biological or chemical agent that exerts a modulating effect of human homeobox gene VentX.
In certain preferred embodiments, the modulating effect comprises an inhibiting effect for treatment of inflammatory autoimmnune conditions
In certain preferred embodiments, the modulating effect comprises that of an immunosuppressant selected from corticosteroid, 6-MP, methotraxate, cellcept and aziothropine.
In certain preferred embodiments, the modulating effect comprises an activating effect comprising that of chemotherapy agents selected from 5-FU, Retinoid acid and DOX.
In certain preferred embodiments, the biological or chemical agent is a polypeptide.
In certain preferred embodiments, the biological or chemical agent is an antibody.
In certain preferred embodiments, the biological or chemical agent is an oligonucleotide.
In another aspect, the invention generally relates to a method for identifying a compound which regulates the VentX expression. The method includes: (a) providing a cell comprising a polynuceotide comprising VentX promoter; (b) contacting the cell with a candidate compound; and (c) measuring the activity of VentX expression.
In certain preferred embodiments, the VentX promoter sequence is of 2.8 KB, cloned with the primers: 5′-CAGCCGAGTCTCACTCTGTC-3′ (SEQ ID NO:1) and 5′-CAAAGCTGGAGAGCTGCTGC-3′ (SEQ ID NO:2), wherein the promoter sequence is placed in front of a luciferase gene to create a construct for a promoter-luciferase assay.
In certain preferred embodiments, the compound is a small molecule agent. In certain preferred embodiments, the compound is a polypeptide. In certain preferred embodiments, the compound is an oligonucleotide.
VentX Expression is Up-Regulated During Monocyte to Macrophage Differentiation
Tissue expression profiling showed that VentX is expressed in monocytes (20). To explore the potential role of VentX in monocyte to macrophage differentiation, VentX expression in peripheral blood monocytes was examined from six healthy donors by RT-PCR. It was found that VentX expression was relatively constant among different individuals (
VentX is Required for Human Primary Monocyte to Macrophage Differentiation
The increased expression of VentX during monocyte to macrophage differentiation suggests a potential role for VentX during the process. To address this hypothesis, small interfering RNA (siRNA) technology was employed to knock down VentX expression in primary monocytes. The efficacy of siVentX was determined by quantitative PCR and western blot analysis (
VentX Promotes Macrophage Differentiation of U937 Cells
U937 is a promonocytic cell line that has been widely used as a model to study monocyte to macrophage differentiation. It has been shown that ectopic expression of several transcription factors, such as BLIMP-1 and IRF-7, was able to trigger macrophage differentiation in U937. (Lu, et al. 2001 J Biol Chem 276:45491-45496; Chang, et al. 2000 Nat Immunol 1:169-176.) To test whether VentX is a bona fide key regulator of monocyte to macrophage differentiation, stable U937 cell lines was generated expressing GFP or GFP.VentX under the control of doxycycline (DOX)-inducible promoter. As shown in
VentX Controls the Expression of M-CSF Receptor
Multiple signaling pathways and transcription factors have been implicated in monocyte differentiation. (Friedman 2007 Oncogene 26:6816-6828.) To identify potential targets of VentX during monocyte differentiation, HL60 cells were ectopically expressed with VentX and treated with phorbol myristate acetate (PMA) to induce monocytic differentiation. (Rovera, et al. 1979 Proc Natl Acad Sci USA 76:2779-2783.) As shown in
Previous studies showed that the M-CSFR promoter is subjected to the regulation of multiple other transcriptional factors such as Foxp1, PU.1, AML1 and C/EBP. (Bonifer, et al. 2008 Front Biosci 13:549-560.) In particular, Foxp1 binds to the forkhead binding sites within M-CSFR promoter and operates as a transcriptional repressor of M-CSFR expression, which raised a possibility that VentX may promote M-CSFR expression through displacement of Foxp1 occupancy on the M-CSFR promoter. (Shi, et al. 2004 J Clin Invest 114:408-418.) To test this hypothesis, ChIP assays were performed to detect the interaction between Foxp1 and the M-CSFR promoter in U937 cells with ectopic expression of VentX and in primary monocytes with knockdown of VentX. As shown in
VentX is Required for the Pro-Inflammatory Response in Macrophages
Macrophages retain a high level of VentX expression (
Macrophages classically activated in vitro by IFN-γ and LPS stimulation display elevated expression of MHC-II and co-stimulatory molecules CD80 and CD86, secrete high level of pro-inflammatory cytokines, and exhibit enhanced antimicrobial activity. (Martinez, et al. 2008 Front Biosci 13:453-461; Mosser, et al. 2008 Nat Rev Immunol 8:958-969.) To investigate whether VentX is required for classical activation of macrophages, a loss-of-function approach by knockdown of VentX was used. First, it was found that surface expression of CD40, CD80 and CD86, but not the HLA-DR, was significantly down-regulated in macrophages transfected with siVentX (
TABLE 1
Effects of VentX knockdown on cytokine mRNA
expression in macrophages
siGFP
siVentX
p Value
TNF-α
0.46 ± 0.04
0.18 ± 0.01
<0.01
IL6
3.40 ± 0.21
1.78 ± 0.14
<0.01
M-SCSF
0.40 ± 0.01
0.14 ± 0.01
<0.01
IL12p35
1.77 ± 0.21
0.37 ± 0.14
<0.01
IL12p40
0.52 ± 0.02
0.61 ± 0.05
>0.05
IL8
471 ± 10.2
172 ± 5.8
<0.01
IL1-β
51.08 ± 3.2
21.93 ± 1.8
<0.01
Also investigated was whether VentX regulates the macrophage alternative activation following IL4 treatment by analyzing several alternative activation markers such as CD163, MR (mannose receptor), AMAC1 (alternative macrophage activation-associated CC chemokine 1; also called CCL-18) and IL10. (Gordon 2003 Nat Rev Immunol 3:23-35; Bouhlel, et al. 2007 Cell Metab 6:137-143.) As shown in
VentX Targets Multiple Pathways to Regulate the Pro-Inflammatory Response in Macrophage
LPS and IFN-γ are two key stimuli to trigger the pro-inflammatory response of macrophages (2, 3). (Martinez, et al. 2008 Front Biosci 13:453-461; Mosser, et al. 2008 Nat Rev Immunol 8:958-969.) Therefore, also investigated was whether VentX affected the signaling pathways of these two stimulants. First examined was whether VentX affected the expression of membrane receptors for LPS and IFN-γ. Using the U937 cell model, it was found that ectopic expression of VentX significantly increased mRNA level of the LPS receptor components (TLR4, CD14, MD-2) and IFN-γ receptors (R1 and R2) (
The TLR4 pathway is coupled to the activation of cytoplasmic transcription factors such as NF-κB and AP-1, which translocate to the nucleus and trigger profound changes in macrophage gene expression. (Schroder, et al. 2006 Immunobiology 211:511-524; Aderem, et al. 2000 Nature 406:782-787.) The IFN-γ signaling is largely mediated by the latent cytosolic factor Stat1 (signal transducer and activator of transcription-1) that is activated during IFN-γ dependent Jak-Stat pathway. (Schroder, et al. 2006 Immunobiology 211:511-524; Hu, et al. 2007 J Leukoc Biol 82:237-243; Schroder, et al. 2004 J Leukoc Biol 75:163-189.) Thus, whether VentX targeted components of TLR4 and IFN-γ downstream signaling pathways was examined. As shown in
Multiple other transcriptional factors are also implicated in macrophage activation by previous studies. For example, Kruppel-like factor (KLF4) and hypoxia-inducible factor (HIF-1) are suggested to promote macrophage activation, whereas KLF2 and Stat6 are inhibitory. (Feinberg, et al. 2005 J Biol Chem 280:38247-38258; Das, et al. 2006 Proc Natl Acad Sci USA 103:6653-6658; Cao, et al. 2010 Blood 116:4404-4414; Kawanami, et al. 2009 J Biol Chem 284:20522-20530; Lentsch, et al. 2001 J Clin Invest 108:1475-1482; Murdoch, et al. 2005 J Immunol 175:6257-6263.) In addition, emerging evidence has suggested the involvement of the transcriptional co-activator p300/CBP in the activation of macrophages. Whether VentX regulates the expression of these factors was examined. As shown in
Expression of VentX and Pro-Inflammatory Cytokines Correlates in Clinical Patients
The findings that VentX controls pro-inflammatory responses in U937 cells (
Immunosuppressants remain as a main therapeutic modality to control inflammation in SLE/RA patients. To determine whether VentX may serve as a therapeutic target of immunosuppressant treatment, the SLE/RA patients were divided into two subgroups: those on immunosuppressant and those on non-immunosuppressant regiments. As shown in
Monocytes Isolation and Culture
Peripheral blood mononuclear cells (PBMC) from healthy adult donors at Children's Hospital Boston were isolated by Ficoll density gradient centrifugation. Experiments with human materials were performed in accordance with guidelines approved by the institutional review committee of Brigham and Women's Hospital. CD14+ monocytes were purified from PBMCs using anti-CD14-coated microbeads (Miltenyi Biotec). The purity of freshly isolated CD14+ monocytes was more than 95% as analyzed by flow cytometry. Monocytes were cultured in 12-well plates at 1×106 cells/ml with RPMI 1640 medium containing 10% fetal bovine serum (FBS). M-CSF, GM-CSF, and IL3 were purchased from PeproTech and used at the final concentration of 100 ng/ml. Cytokines were added to cultures every 2 or 3 days.
RNA Interference
Human primary monocytes were transfected using the Human Monocyte Nucleofector Kit (Lonza) according to the manufacturer's instructions. Briefly, 5×106monocytes were resuspended into 100 μl nucleofector solution with 0.5 nmol of either VentX siRNA (forward: 5′-UUCAGAAUCGCCGCAUGAAACACAAACGG-3′ (SEQ ID NO:4); reverse: 5′-CCGUUUGUGUUUCAUGCGGCGAUUCUGAA-3′ (SEQ ID NO:5)) or non-effective GFP siRNA (forward: 5′-UGACCACCCUGACCUACGGCGUGCAGUGC-3′ (SEQ ID NO:6); 5′-reverse: GCACUGCACGCCGUAGGUCAGGGUGGUCA-3′ (SEQ ID NO:7)) before electroporation with nucleofector II Device (Lonza). Cells were then immediately removed from the device and incubated overnight with 1 ml pre-warmed Human Monocyte Nucleofector Medium containing 2 mM glutamine and 10% FBS. Cells were then resuspended into complete RPMI medium and treated with appropriate cytokines to induce differentiation into macrophages. Similarly, macrophages derived from monocytes were transfected with Human Macrophage Nucleofector Kit (Lonza) following the manufacturer's instructions.
Generation of U937 Cell Line Conditionally Expressing VentX
Human promonocytic cell line U937 was obtained from American Type Culture Collection (ATCC). Plasmid constructs expressing VentX have been described previously (20). GFP.VentX fusion fragment was cut from pCS2 expression vector through digestion with BamHI/SnaBI and subcloned into pRetroX-Tight-Puro retroviral vector (Retro-X™ Tet-On Advanced Expression System, Clontech) digested with BamHI/NruI. Retroviruses were packaged through co-transfection of pCL-Ampho packaging vector (IMGENEX) and retroviral vectors into HEK293 cells. A U937 cell line conditionally expressing GFP.VentX was generated through co-transduction of pRetroX-GFP.VentX and pRetroX-Tet-On Advanced retroviruses. GFP.VentX positive cells were sorted by FACSAria high-speed sorter (BD Bioscience) after incubation with 1.0 μg/ml doxycycline for 24 hours (Dana-Farber Cancer Institute Flow Cytometry Core Facility). Sorted cells were then maintained in RPMI 1640 medium in the absence of doxycycline. A U937 cell line conditionally expressing GFP was similarly generated for comparison.
FACS Analysis
Phenotypic analysis of monocytes/macrophages was performed using flow cytometry after immunolabeling of cells with fluorescence dye conjugated antibodies. The following antibodies were used: PE-conjugated anti-CD71, CD11b, CD11c, CD16, CD64, CD80, CD86, HLA-DR, CD14, TLR4, IL1-β and TNF-α, and FITC-conjugated anti-CD40, CD36 (eBioscience); FITC-conjugated anti-mannose receptor (MR), and unconjugated mouse anti-MCSFR (R & D Systems). Isotope control labeling was performed in parallel. Antibodies were diluted as recommended by the supplier. PE-conjugated rabbit against mouse IgG antibody was used for secondary M-CSFR staining Labeled cells were analyzed with FACScan flow cytometer (BD Bioscience) using CellQuest software. Results are expressed as the percentage of positive cells and/or mean fluorescence intensity (MFI) values after subtraction of the MFI obtained with the isotype control antibody.
Western Blot
Cells were lysed in solution A (50 mM Tris-HCl, pH 7.8, 420 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.34 M sucrose, 10% glycerol, 1 mM Na3VO4, 10 mM NaF and β-glycerophosphate, 1 mM PMSF, and protease inhibitor cocktail) as described previously (64). Proteins resolved by SDS-PAGE were transferred onto PVDF membrane, which were detected with optimal dilutions of primary antibodies, followed by horseradish peroxidase-linked secondary antibodies. Primary antibodies used were from Cell Signaling except anti-VentX sera.
RT-PCR
Total RNA was isolated by the TRIzol reagent, and an equal amount of RNA was used for first-strand cDNA synthesis with SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol. To amplify VentX cDNA with conventional PCR, AccuPrime™ Taq DNA polymerase system (Invitrogen) was used following the manufacturer's instructions. PCR products were separated on 2% agarose gels and stained with ethidium bromide. GAPDH was used as an internal control. Quantitative measurement of VentX and cytokines cDNA were performed with SYBR Green on a LightCycler® (480 Real-Time PCR System; Roche). The primers used are listed in the Sequence Listing (SEQ ID NOs: 24-109).
Cytokine Measurements
Levels of IL-1β and TNF-α and IL12p70 in the supernatants of E. coli LPS (Sigma) and IFN-γ (PeproTech) treated macrophage or LPS treated U937 cells were quantified using ELISA kits obtained from eBiosciences. Analyses were conducted according to the manufacturer's instructions.
Phagocytosis Assay
Phagocytosis of cultured monocytes/macrophages or U937 cells were performed with pHrodo™ S. aureus BioParticles® conjugates from Invitrogen. Briefly, S. aureus particles were first sonicated to achieve homogeneous solution and then opsonized for 60 min at 37° C. with opsonizing reagent obtained from Invitrogen (Cat. S-2860). After the S. aureus particles were washed 3 times in PBS, they were resuspended to an appropriate concentration for phagocytosis assay. 5×105 cells were incubated with 100 μl of opsonized particles for 2 hours at 37° C. After extensive washings, cellular phagocytosis of bioparticles was monitored by flow cytometry. Negative controls were also performed in parallel by incubating cells with particles on ice instead of at 37° C.
Detection of Reactive Oxygen Species (ROS) and Nitric Oxide (NO)
The ROS level in activated macrophages was detected with Image-iT® LIVE Green Reactive Oxygen Species Detection Kit (Invitrogen) basically following the manufacturer's instructions except that the results were analyzed by both fluorescence microscope and flow cytometry. The NO level was determined by Griess Reagent Kit for Nitrite Determination (Invitrogen) following the protocol provided by the manufacturer.
Cytostaining
For Wright-Giemsa staining, a staining kit from Sigma was used according to the manufacturer's instructions.
Mixed Lymphocyte Reaction
Macrophages were generated by incubating CD14-beads isolated monocytes with 100 ng/ml M-CSF for 4 days. Macrophages were then transfected with siRNA against VentX or GFP, as described above. After 3 days of transfection, the cells were harvested, washed 3 times in PBS, and irradiated (5000 rad) before incubation with allogenic naive CD4+ T cells (105/well) for 7 days in 96-well flat-bottom microplates (Costar). Various numbers of irradiated macrophages were added as indicated. The cells were pulsed for the last 18 hours with 1 μCi of [3H] thymidine to determine T cell proliferation.
Luciferase Reporter Assay
The 1.56 kb fragment of M-CSFR promoter region was amplified with forward primer: 5′-GTTACGCGTGGGAAGCCAAGGTATGAGAATC-3′ (SEQ ID NO:8), and reverse primer: 5′-AAGCTCGAGCCTCGGTGGGGAAGTGGCAG-3′ (SEQ ID NO:9). The 2.8 kb fragment of VentX promoter region was amplified with forward primer: 5′-CAGCCGAGTCTCACTCTGTC-3′ (SEQ ID NO:1), and reverse primer: 5′-CAAAGCTGGAGAGCTGCTGC-3′ (SEQ ID NO:2). The PCR product was subsequently cloned into pGL3 luciferase reporter. 500 ng reporter plasmid with 500 ng of pcDNA-VentX plasmid or empty pcDNA vector were transfected into U937 cells or primary monocytes through electroporation. 10 ng Renilla luciferase plasmid was included for each transfection to normalize reporter activity. Cells were harvested at 48 hours after transfection and analyzed with Dual-Luciferase Reporter Assay System (Promega).
Gel Shift Assay
VentX protein was generated with a in vitro translation kit (TNT® Coupled Reticulocyte
Lysate Systems, Promega). The pCS2-VentX plasmid with SP6 promoter was used as template and translated VentX protein was verified by western blot analysis. Gel shift assay was performed with a fluorescence-based Electrophoretic Mobility Shift Assay (EMSA) Kit from Invitrogen following the manufacturer's instruction. The following double-stranded oligonucleotides were used in the experiments: 5′-CTGCGTCTCTAAAATAATAATAATAAATTTTTAAAAGATATGC-3′(SEQ ID NO:12)(wild type M-CSFR probe, putative homeodomain binding sequence is in bold and underlined); 5′-CTGCGTCTCTAAAATAAGAAGAATAAATTTTTAAAAGATATGC-3′(mutant M-CSFR)(SEQ ID NO:13).
ChIP Assay
U937 cell lines conditionally expressing GFP or GFP. VentX were employed to detect the potential interaction of VentX with M-CSFR, Stat1 and JunB promoters. Cells were treated with 1.0 μg/ml doxycycline for 2 days and harvested for chromatin immunoprecipitation (ChIP) assay. The ChIP procedure was performed with a kit from Upstate Biotechnology (Billerica, MA) following the manufacturer's instructions. The GFP antibody (Santa Cruz Biotechnology) was used for the immunoprecipitation. M-CSFR promoter region containing a putative homeodomain binding site was amplified with specific primers: 5′-TAGAGATAACGTCAGATCTCAC-3′ (SEQ ID NO:14) and 5′-CAAAGAGAAGTTAGGTTGCATG-3′ (SEQ ID NO:15); the STAT1 promoter was amplified with primers: 5′-TGACTGATGGAAAGGGGTGG-3′ (SEQ ID NO:16) and 5′-GCCACCTGTTCTTGGGAGAT-3′ (SEQ ID NO:17); the JunB promoter was amplified with primers: 5′-GCTTACTAGCTTTCTGCATA-3′ (SEQ ID NO :18) and 5′-GGAGGGGAGAGATCAAAAGG-3′ (SEQ ID NO:19); the constant region of immunoglobulin M heavy chain gene (Cμ), which serves as a negative control, was amplified with the following primers: 5′-AACCCTTTTCCCCCTCGTCT-3′ (SEQ ID NO:20) and 5′-AGCACCTGTGAGGTGGCTGC-3′ (SEQ ID NO:21). To detect if VentX competes with Foxp1 to bind to M-CSFR promoter region, U937 cells were treated with 1.0 μg/ml doxycycline for 2 days as described above, or primary monocytes were transfected by electroporation with siGFP or siVentX. Cell lysates were then immunoprecipitated with Foxp 1 antibody (Cell signaling) and the M-CSFR promoter region containing the Foxp 1 binding site was amplified with primers: 5′-GCTTTAGAAGGGCCCCAAAC-3′ (SEQ ID NO:22) and 5′-CTACTAGCTCCGCAGGGATC-3′ (SEQ ID NO:23). All PCR products were separated on 8% polyacrylamide gel and visualized by ethidium bromide staining. ps Statistical Analysis
Data were analyzed using the paired Student's t test (2-tailed) and Wilcoxon rank-sum test. The differences with p value <0.05 were considered statistically significant.
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance which can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
Sequence listings and related materials in the ASCII text file named “ZLZ-001US_SEQ-2017-0614_ST25.txt” and created on Jun. 14, 2017 with a size of about 28 kilobytes, is hereby incorporated by reference.
| Patent | Priority | Assignee | Title |
| Patent | Priority | Assignee | Title |
| 7994126, | Feb 22 2006 | Zhenglun, Zhu; Hong, Gao | Treatment of cellular proliferative disorders with compositions comprising Hom polypeptide (also known as Xom, VENTX2) |
| WO2007101063, | |||
| WO2011008947, |
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